Application of kappa opioid receptor agonists in the preparation of drugs for preventing and / or treating temporal lobe epilepsy

Abstract

The present invention provides an application of a kappa opioid receptor agonist in the preparation of a drug for preventing and / or treating temporal lobe epilepsy, wherein the kappa opioid receptor agonist is a non-nitrogen-containing diterpenoid substance, and the non-nitrogen-containing diterpenoid substance is carnosine A. The present invention uses carnosine A to inhibit the excitability mechanism in the brain through kappa opioid receptors to prevent and treat temporal lobe epilepsy; compared with other existing anti-epileptic drugs, carnosine A acts on a completely new target, can simultaneously regulate multiple ion channels located at different positions, has a stable anti-epileptic effect, and is not prone to drug resistance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a kappa opioid receptor agonist in the preparation of a drug for preventing and / or treating temporal lobe epilepsy. Background Art

[0002] The origin of temporal lobe epilepsy is located in the bilateral temporal lobes of the brain, and the number of patients accounts for about 70% of all epilepsy patients. There are currently two main types of drugs used clinically to treat temporal lobe epilepsy, one is ion channel blockers represented by carbamazepine, and the other is enhancers that enhance the activity of the GABA system represented by sodium valproate. However, the above-mentioned drugs in the prior art have many shortcomings, such as slow onset, ineffectiveness for about 30% of epilepsy patients, and serious neurological and somatic adverse reactions after long-term use.

[0003] The onset of temporal lobe epilepsy is closely related to hippocampal sclerosis. During epileptic discharges, the excitability of hippocampal neurons increases sharply, the oxygen demand increases significantly, the hippocampal tissue is ischemic and hypoxic, the local inflammatory response intensifies, causing neuronal necrosis accompanied by an increase in glial cells, and then inducing hippocampal sclerosis. The loss of GABAergic interneurons during hippocampal sclerosis reduces the inhibition of glutamatergic neurons and enhances the excitatory mechanism in the brain, thereby exacerbating epileptic discharges. Therefore, hippocampal sclerosis is both a consequence of refractory or recurrent epilepsy and a cause of epileptic seizures. Reducing the hippocampal excitability mechanism and subsequent series of reactions is an effective measure to prevent hippocampal sclerosis and the pathological process of epilepsy.

[0004] On the other hand, the kappa opioid receptor (KOR) known in the prior art is widely distributed in the hippocampus and is expressed in various types of neurons and glial cells such as granule cells and pyramidal cells. Dynorphin is an endogenous ligand of the kappa opioid receptor. It acts on the kappa opioid receptors of the presynaptic and postsynaptic membranes in the form of co-release with glutamate in the hippocampal glutamatergic synaptic transmission. The former inhibits Ca 2+ Channel opening reduces Ca 2+ The influx reduces the excitability of the presynaptic neuron and prevents further release of glutamate; the latter increases K + Channel-mediated outward K +The current stabilizes the postsynaptic membrane and avoids overexcitation of the postsynaptic neurons. Therefore, the κ opioid receptor can be used as a potential drug target for the treatment of temporal lobe epilepsy. However, the κ opioid receptor agonists in the prior art are mainly divided into peptide substances based on the dynorphin structure, nitrogen-containing small molecule compounds, and non-nitrogen-containing diterpenoid compounds based on the molecular skeleton of the natural compound carnosine A. The first two agonists cannot be used clinically due to different reasons such as inability to penetrate the blood-brain barrier, low bioavailability, and serious side effects. Carnosine A is the active component of Mexican sage (Salvia divinorum), a perennial herbaceous plant of the Lamiaceae (mint) family native to Mexico. Carnosine A is the most highly effective, naturally occurring non-peptide and the only non-nitrogen-containing κ opioid receptor agonist; in particular, carnosine A is a very hydrophobic molecule and is insoluble in water. It is known to be soluble in organic solvents (such as ethanol, DMSO and acetone), but these solvents are not suitable for routine clinical use, especially for intravenous (IV) delivery. On the other hand, in the prior art, caryophyllin A is mainly used clinically for neurological diseases. It is usually taken orally or inhaled to achieve effective blood concentrations and acts on the target brain area through the blood-brain barrier. However, there are currently no reports on the use of non-nitrogen-containing diterpenoid κ opioid receptor agonists as drugs for the prevention and / or treatment of temporal lobe epilepsy. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention discloses the use of a non-nitrogen-containing diterpenoid κ opioid receptor agonist in the preparation of a drug for preventing and / or treating temporal lobe epilepsy.

[0006] To achieve the above object, the present invention provides a use of a κ opioid receptor agonist in the preparation of a drug for preventing and / or treating temporal lobe epilepsy, wherein the κ opioid receptor agonist is a non-nitrogen-containing diterpenoid substance.

[0007] Preferably, the non-nitrogen-containing diterpenoid substance is salvinoresin A, which is a non-nitrogen-containing diterpenoid substance extracted from salvia, and is a natural selective κ opioid receptor agonist. Its chemical name is 2H-naphthalene (2,1-C) pyran-7-carboxylic acid, 9-(acetyloxy)-2-(3-furyl) dodecyl-6A, 10B-dimethyl-4,10-dioxy-methyl ester-(2S, 4AR, 6AR, 7R, 9S, 10AS, 10BR)-two-dimensional guanosine A, and its chemical structure is shown in formula (1):

[0008]

[0009] Preferably, caryophyllin A can prolong the latency period of clonic seizures, reduce the duration and intensity of epileptic seizures, reduce the loss of neurons in the hippocampus, and reduce the level of inflammatory mediators in the hippocampus in the prevention and / or treatment of temporal lobe epilepsy.

[0010] Preferably, the drug for preventing and / or treating temporal lobe epilepsy targets the kappa opioid receptor agonist.

[0011] Preferably, the drug for preventing and / or treating temporal lobe epilepsy comprises at least one of an oral agent, an inhalation agent and an injection.

[0012] Preferably, the injection comprises carnosine A dissolved in physiological saline containing 5% dimethyl sulfoxide.

[0013] Preferably, in the mouse experiment, the intake dose of caryophyllin A is 0.3-1.0 mg / kg, which can reduce the protein levels of pro-inflammatory factors IL-1β and TNF-a, reduce and increase the protein level of iNOS, and increase the protein levels of Arg-1.

[0014] Preferably, when the intake dose of caryophyllin A is 1.0 mg / kg, the total duration of epileptic seizures within 3 hours can be reduced.

[0015] The present invention is based on a temporal lobe epilepsy model mouse induced by intraperitoneal injection of pilocarpine, and adopts a variety of modern neuroscience techniques such as electroencephalography, immunohistochemistry, molecular biology, and behavior to study the anti-epileptic effect and related mechanism of a non-nitrogen-containing diterpenoid κ opioid receptor agonist caryophyllin A, so as to enable it to become a clinical drug for the prevention and treatment of temporal lobe epilepsy.

[0016] In a specific embodiment of the present invention, the injection method is used as an example for detailed description. Carnosine A is dissolved in physiological saline containing 5% dimethyl sulfoxide and given to mice with epilepsy model caused by pilocarpine by intraperitoneal injection. The experiment found that carnosine A can prolong the latency of clonic seizures, reduce the time and intensity of epileptic seizures, reduce the loss of neurons in the hippocampus, and reduce the level of inflammatory mediators in the hippocampus, and has a neuroprotective effect on epilepsy model mice. Based on the test conducted by injection as an example, carnosine A is used in drugs for the prevention and / or treatment of temporal lobe epilepsy, which has a significant effect, a stable anti-epileptic effect, and is not easy to produce drug resistance.

[0017] The technical effects produced by the present invention are:

[0018] 1. The present invention uses carnosine A to simultaneously reduce the excitability of presynaptic cells and postsynaptic cells through κ opioid receptors located in different synaptic structures, thereby inhibiting the excitability mechanism in the brain and preventing and treating temporal lobe epilepsy; compared with other existing anti-epileptic drugs, carnosine A acts on a completely new target and can simultaneously regulate multiple ion channels located in different positions, with a stable anti-epileptic effect and is not prone to drug resistance.

[0019] 2. Based on the temporal lobe epilepsy model mice induced by intraperitoneal injection of pilocarpine, the present invention uses a variety of modern neuroscience techniques such as electroencephalography, immunohistochemistry, molecular biology, and behavior to study the anti-epileptic effects and related mechanisms of the non-nitrogen-containing diterpenoid κ opioid receptor agonist caryophyllin A, and finds that it is expected to become a clinical drug for the prevention and treatment of temporal lobe epilepsy.

[0020] 3. The caryophyllin A used in the present invention as a κ opioid receptor agonist can exert anti-inflammatory and anti-apoptotic effects after intraperitoneal injection, effectively reduce the loss of hippocampal neurons caused by excessive neuronal excitation, and slow down the process of hippocampal sclerosis.

[0021] 4. The present invention applies carnosine A to drugs for treating epilepsy. It can be taken into the blood through various ways such as oral administration, inhalation, injection, etc., and can easily pass through the blood-brain barrier, and has strong drugability. At an effective anti-epileptic dose, carnosine A has small peripheral side effects and has an antidepressant effect in the central nervous system, which can avoid the side effect of existing anti-epileptic drugs that aggravates depressive symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1a-1b They are respectively diagrams of behavioral test results after pilocarpine modeling in a typical embodiment of the present invention.

[0023] Figure 2a-2d This is a diagram of EEG detection results in a typical implementation scheme of the present invention.

[0024] Figure 3 This is a HE staining image of the hippocampus in a typical embodiment of the present invention.

[0025] Figure 4a-4d This is a graph showing the detection results of inflammatory mediators in the hippocampus in a typical embodiment of the present invention.

[0026] Figure 5a-5g This is a diagram showing the results of detecting the phenotype of microglia in the hippocampus in a typical embodiment of the present invention.

[0027] Figure 6a-6c This is a graph showing the detection results of apoptosis-related proteins in the hippocampus in a typical embodiment of the present invention. DETAILED DESCRIPTION

[0028] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.

[0029] The present invention provides an application of a kappa opioid receptor agonist in the preparation of a drug for preventing and / or treating temporal lobe epilepsy. The kappa opioid receptor agonist is a non-nitrogen-containing diterpenoid substance.

[0030] Specifically, the non-nitrogen-containing diterpenoid substance is caryophyllein A.

[0031] Salvinin A is a non-nitrogenous diterpenoid extracted from sage. It is a natural selective κ opioid receptor agonist.

[0032] In the prevention and / or treatment of temporal lobe epilepsy, caryophyllein A can prolong the latency period of clonic seizures, reduce the duration and intensity of epileptic seizures, reduce the loss of neurons in the hippocampus, and reduce the level of inflammatory mediators in the hippocampus.

[0033] In the drug for preventing and / or treating temporal lobe epilepsy, the kappa opioid receptor is used as a target.

[0034] The drug for preventing and / or treating temporal lobe epilepsy includes at least one of an oral agent, an inhalation agent and an injection.

[0035] As a preferred embodiment, the injection comprises dissolving caryophyllin A in physiological saline containing 5% dimethyl sulfoxide.

[0036] The sources of experimental materials required in the present invention include:

[0037] Carnosin A: purchased from TargetMol (cas#83729-01-5).

[0038] The experimental mice were purchased from Cavens Laboratory Animal Co., Ltd. and housed in the Experimental Animal Center of Xuzhou Medical University.

[0039] EEG recording instrument: from LabChart system.

[0040] Stereotaxic instrument: from Rayward Company.

[0041] Main reagents: Scopolamine and pilocarpine were purchased from GLPBIO, diazepam was purchased from Tianjin Jinyao Pharmaceutical Co., Ltd.

[0042] TNF-a, Il-1b enzyme-linked immunosorbent assay kits: purchased from Ruixin Biotechnology Co., Ltd.

[0043] Unless otherwise specified, the chemical reagents used in the present invention are all commercially available.

[0044] The technical scheme of the present invention is further described in detail below through specific examples. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0045] Example 1

[0046] Build a test model

[0047] This embodiment provides a control group, an epilepsy-induced group, a low-dose group and a high-dose group, specifically including:

[0048] 1. Prepare caryophyllin A solution:

[0049] Dissolve 1 mg of Salvinorin A (SA) in 2 ml of physiological saline containing 5% dimethyl sulfoxide for later use, wherein the concentration of Salvinorin A is 0.5 mg / mL. For intraperitoneal injection, use 5% dimethyl sulfoxide and dilute it to 0.10 mg / mL.

[0050] 2. Establish medication groups:

[0051] Forty 8-week-old C57 mice were randomly selected according to their body weight and divided into four groups: control group (Control), pilocarpine-induced epilepsy group (SE group or pilocarpine group), pilocarpine + safollicularis A low-dose group (SE+SA0.3 or, SA low-dose group) and pilocarpine + safollicularis A high-dose group (SE+SA 1.0 or, SA high-dose group), with 10 mice in each group.

[0052] In this example, 2 weeks before the pilocarpine modeling, the mice in each group were intraperitoneally injected with the preventive medication, and the preventive medication lasted for 14 days.

[0053] Specifically, each group of mice was intraperitoneally injected daily, wherein the SA low-dose group and the SA high-dose group were intraperitoneally injected with SA solution, respectively; the SA dose injected in the SA low-dose group was 0.3 mg / kg (for example, 0.075 mL of SA solution was intraperitoneally injected into a 25 g body weight mouse), and the SA dose injected in the SA high-dose group was 1.0 mg / kg (for example, 0.25 mL of SA solution was intraperitoneally injected into a 25 g body weight mouse); the control group and the pilocarpine group were both injected with 5% dimethyl sulfoxide-containing saline as a control (for example, 0.25 mL of 5% dimethyl sulfoxide-containing saline was intraperitoneally injected into a 25 g body weight mouse).

[0054] 3. Build the test model:

[0055] On the day of modeling (the 15th day), the other three groups except the control group were intraperitoneally injected with scopolamine (2 mg / kg) to inhibit the peripheral effects of pilocarpine; half an hour later, the other three groups except the control group were intraperitoneally injected with pilocarpine (320 mg / kg), and the control group was injected with normal saline containing 5% dimethyl sulfoxide.

[0056] After the injection, the mice were transferred to a cage with a constant temperature of 28-30 degrees Celsius to observe their behavioral changes for the next 3 hours.

[0057] The experiment was terminated 3 hours later by intraperitoneal injection of diazepam (10 mg / kg) and 1 mL of 5% glucose solution was intraperitoneally injected to increase the survival rate of mice, and then the mice were transferred back to the home cage.

[0058] Within one week after modeling, the mice in the SA high-dose group and the SA low-dose group were injected intraperitoneally every day, and the injection dose was the same as in 2. The control group and the SE group were injected with normal saline containing 5% dimethyl sulfoxide, and the injection dose was the same each time.

[0059] Example 2

[0060] Based on the model and drug administration grouping provided in Example 1, a pharmacological experiment was performed. The experimental detection indicators in this example include:

[0061] 1. Detection of carnosine A improving pilocarpine-induced epileptic seizure behavior in mice

[0062] After pilocarpine injection, the mice were transferred to a cage with a constant temperature of 28-30 degrees Celsius and their behavioral changes were observed for the next 3 hours.

[0063] According to the Racine scoring principle, the seizures of mice were divided into 5 levels: Level 1: lip and facial twitching; Level 2: nodding; Level 3: forelimb spasms; Level 4: standing; Level 5: standing and falling, whole body spasms. If the observation level reaches level 3 or above, it can be regarded as an epileptic seizure. The time to the first seizure is the seizure latency. The latency period and the number and total time of seizures within 3 hours are recorded.

[0064] Test results: See Figure 1a and Figure 1b ,Depend on Figure 1a It can be seen that the onset latency duration of the SA low-dose group (SE+SA0.3) and the SA high-dose group (SE+SA 1.0) was significantly increased compared with the SE control group.

[0065] Depend on Figure 1b It can be seen that 1.0 mg / kg SA significantly reduced the total duration of epileptic seizures within 3 hours.

[0066] 2. Carnosine A improves the detection of pilocarpine-induced cortical epileptiform discharges in mice

[0067] Electrodes were implanted in the cortex of mice in each group on the third day after pilocarpine injection, and cortical EEG (electroencephalogram) was measured after the animals recovered for one week.

[0068] The EEG electrodes were implanted in the prefrontal cortex, including the anterior fontanelle: anterior 2.0 mm, mediolateral 2.0 mm, and dorsoventral 0.5 mm; the reference electrode was placed above the cerebellar skull.

[0069] The frequency of epileptic seizures in each group of mice within one day, the maximum abundance of EEG and the maximum power in the spectrum during epileptic seizures were recorded.

[0070] The test results can be found in Figure 2a-2d ,Depend on Figure 2b It can be seen that the number of seizures per day in the SA low-dose group (SE+SA0.3) and the SA high-dose group (SE+SA 1.0) was significantly reduced compared with the SE control group.

[0071] Depend on Figure 2a , Figure 2c and Figure 2d It can be seen that 1.0 mg / kg SA significantly reduced the maximum abundance of seizure discharges ( Figure 2a , Figure 2c ) and maximum power ( Figure 2a , Figure 2d ).

[0072] 3. Test on the effect of carnosine A on improving pilocarpine-induced hippocampal neuron loss in mice

[0073] One week after the injection of pilocarpine, the mice were killed, and coronal sections of the mouse brain tissue were taken. Section samples with complete hippocampal structure were taken for HE staining, and observed and photographed under a microscope.

[0074] The test results from Figure 3 As shown, the dentate gyrus (DG) and CA3 regions of the hippocampus of mice with pilocarpine-induced epilepsy model showed significant multicellular shrinkage and cell death, while the neuronal death in the hippocampus of mice in the low-dose SA group (SE+SA0.3) and the high-dose SA group (SE+SA1.0) was significantly improved compared with the SE control group.

[0075] 4. Test on the effect of carnosine A on the increase of inflammatory mediators in the hippocampus of mice induced by pilocarpine

[0076] One week after pilocarpine injection, the mice were killed, and the hippocampal tissues were taken to extract RNA and protein, respectively, and RT-PCR and ELISA experiments were performed to detect the mRNA and protein levels of IL-1β and TNF-a in the hippocampal tissues.

[0077] The test results from Figure 4a-4d As shown in the figure, the RT-PCR results showed that both high and low doses of SA significantly reduced the expression of IL-1β ( Figure 4a )、TNF-a( Figure 4b ) mRNA level and TNF-a protein level ( Figure 4c ) increases. At the same time ( Figure 4d ) The IL-1β protein level was significantly reduced in the high-dose SA group, but there was no significant change in the low-dose SA group compared with the SE control group.

[0078] 5. Test on the effect of carnosine A on the improvement of pilocarpine-induced M1 phenotype conversion of mouse hippocampal microglia

[0079] One week after the pilocarpine injection, the mice were killed, and the coronal sections of the mouse brain tissue were taken. The slice samples with complete hippocampal structure were taken for immunofluorescence staining and observed and photographed under a microscope. At the same time, part of the hippocampal tissue was used for protein extraction, followed by immunoblotting experiments.

[0080] Test results: Figure 5a-5f As shown, the immunofluorescence results showed that compared with the SE control group, high-dose SA significantly reduced the number of iNOS-positive cells, namely M1 pro-inflammatory microglia, and increased the number of Arg-1-positive cells, namely M2 anti-inflammatory microglia, in the three subregions of the hippocampus, DG, CA1, and CA3 ( Figure 5a-5e ), the results of immunoblotting echoed the results of immunofluorescence, and the protein levels of iNOS and Arg-1 in the hippocampus of the high-dose SA group were also significantly increased compared with the SE group ( Figure 5f , Figure 5g ).

[0081] 6. Test on the effect of carnosine A on the increase of apoptotic protein levels in the hippocampus of mice induced by pilocarpine

[0082] One week after pilocarpine injection, the mice were killed, and the hippocampal tissues were taken to extract proteins for immunoblotting experiments to detect the levels of apoptosis-related proteins Bcl-2 and caspase-3 in the hippocampal tissues.

[0083] Test results: Figure 6a-6c As shown in the figure, WB results showed that compared with the SE control group, high-dose SA significantly increased the expression of the anti-apoptotic protein Bcl-2 ( Figure 6a , Figure 6b ), reduced the expression of cleaved caspase-3 ( Figure 6a , Figure 6c ); the low-dose SA group reduced the expression of cleaved caspase-3 ( Figure 6a , Figure 6c ).

[0084] The results of the above animal experiments show that carnosine A can act on the κ opioid receptors on neurons in the brain through intraperitoneal injection, inhibit the abnormal excitability of neurons, reduce the number and duration of epileptic seizures in animals, and improve the abnormal behavior caused by epileptic discharges. At the same time, carnosine A can protect neurons through anti-inflammatory and anti-apoptotic effects, effectively reduce the loss of hippocampal neurons caused by excessive neuronal excitation, slow down the process of hippocampal sclerosis, and play an active role in preventing the pathological process of epilepsy and treating epileptic seizures.

[0085] The above are only preferred embodiments of the present invention, which do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Within the spirit and principle of the present invention, changes, modifications, replacements, integrations and parameter changes to these embodiments by conventional substitutions or by being able to achieve the same functions without departing from the principles and spirit of the present invention all fall within the protection scope of the present invention.

Claims

1. Use of a kappa opioid receptor agonist in the preparation of a drug for preventing and / or treating temporal lobe epilepsy, characterized in that: The kappa opioid receptor agonist is a non-nitrogen-containing diterpenoid substance; The non-nitrogen-containing diterpenoid substance is carnosine A, and its chemical formula is shown in Formula 1: (1); In the mouse experiment, adult mice were used as experimental subjects, and the mouse model was established by pilocarpine induction; The concentration of carnosine A solution used for intraperitoneal injection was 0.10 mg / mL; The intake dose of caryophyllein A is 0.3~1.0 mg / kg.

2. The use according to claim 1, characterized in that: In the prevention and / or treatment of temporal lobe epilepsy, caryophyllein A can prolong the latency period of clonic seizures, reduce the duration and intensity of epileptic seizures, reduce the loss of neurons in the hippocampus, and reduce the level of inflammatory mediators in the hippocampus.

3. The use according to claim 1, characterized in that: The drug for preventing and / or treating temporal lobe epilepsy takes the kappa opioid receptor as a target.

4. The use according to claim 1, characterized in that: The drug for preventing and / or treating temporal lobe epilepsy includes at least one of an oral agent, an inhalation agent and an injection.

5. The use according to claim 1, characterized in that: The injection comprises dissolving carnosin A in physiological saline containing 5% dimethyl sulfoxide.

6. The use according to claim 1, characterized in that: In mouse experiments, the intake of 1.0 mg / kg of caryophyllein A can reduce the total duration of epileptic seizures within 3 hours.

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